CN104478981B - The extracting method of (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone in Caulerpa racemosa (Forssk) Web V. Bos - Google Patents
The extracting method of (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone in Caulerpa racemosa (Forssk) Web V. Bos Download PDFInfo
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Abstract
The present invention relates to the extracting method of (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone in a kind of Caulerpa racemosa (Forssk) Web V. Bos。This compound adopts extraction, concentrating under reduced pressure, extraction, silica gel column chromatography and gel column chromatography purification to obtain from Caulerpa racemosa (Forssk) Web V. Bos, its structure measured by physicochemical constant and spectral data analysis and determine。Neuroprotective experiment proof through repeatedly external SH-SY5Y cell injury: this compound is to amyloid-beta 25 35 (A β25-35) the SH-SY5Y cell that damages has significant protective effect, can be used for preparing neuroprotective and preventing Alzheimer disease drug。
Description
Technical field
The present invention relates to a kind of extraction (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone and its production and use from Caulerpa racemosa (Forssk) Web V. Bos；The invention still further relates to this compound for A β25-35The SH-SY5Y cell of damage has the active component of protective effect, thus it can be used for preparing nerve protection medicine and preventing Alzheimer disease drug。
Background technology
Alzheimer's disease (Alzheimer ' sdisease, AD) it is a kind of central nervous system's constitutional degenerative disease, this disease is suffered from more than 35,000,000 people in the whole world。Its clinical manifestation be Progressive symmetric erythrokeratodermia dysmnesia, the dyskinesia, cognitive disorder etc. (Prince, M.eral,WorldAlzheimerReport2013;Alzheimer ' sDiseaseInternational:London, UK, 2013;Pp1?92)。The existing AD patient of China there are about 8,000,000 more than。Owing to end-stage AD patient can't take care of oneself, and it is often accompanied by serious spirit, nervous symptoms, not only seriously reduces the quality of life of old people, return family and society brings heavy spirit and financial burden。AD, as the main Types of senile dementia, is the 4th cause of the death after heart disease, cancer, apoplexy。Therefore, the preventing and treating of AD has been caused the extensive concern of medicine sector or even the whole society。Research for the discovery of the AD pilot compound carried out and the mechanism of action thereof, it has also become Chinese and western medicine pharmacy worker endeavours the important topic of research。
Caulerpa racemosa (Forssk) Web V. BosCaulerparacemosa(Forssk?L) J.Agardh system Chlorophyta (Chlorophyta) Chlorophyceae (Chlorophyceae) Siphonales (Siphonales) Caulerpaceae (Caulerpaceae) Caulerpa belong to (Caulerpa) sea-plant, be distributed mainly on Perenniporia martius marine site, be grown on the rock of below Intertidal zone, coral reef or in, on the sand ground of low tide band。Also have widely distributed in China marine site, focus primarily upon Dongshan, Fujian, Taiwan, Hainan, Xisha, Coast of Guangdong Province。Research both at home and abroad shows, in Caulerpa racemosa (Forssk) Web V. Bos containing the compositions such as sesquiterpenoids, Diterpenes, steroid, glyceride type, aromatics, amide-type and alkaloids (Alarif, W.M.etal,Clean:Soil,Air,Water, 2010,38 (5-6), 548 557；Wang, H.etal,BotanicaMarina,2007,50(3),185–190;Ayyad,S.-E.N.etal,AlexandriaJournalofPharmaceuticalSciences,1994,8(3),217–219;Anjaneyulu,A.S.R.etal,JournalofNaturalProducts, 1992,55 (4), 496 499)。Wherein most terpenoids contain Isosorbide-5-Nitrae-diacetoxy (aldehyde radical) the butadiene structure segment that nature is rare, and alkaloids is then the bis-indole compounds that structure is peculiar。Modern pharmacology research shows, the secondary metabolite found from Caulerpa racemosa (Forssk) Web V. Bos often show very strong antibacterial, antiinflammatory, antiviral, parasite killing, antitumor and poison various biological activity such as fish (Anjaneyulu, A.S.R.etal,Phytochemistry,1991,30(9),3041-3042;Anjaneyulu,A.S.R.etal,JournaloftheIndianChemicalSociety,1991,68(8),480;Capon,R.J.Phytochemistry,1983,22(6),1465-1467;Nielsen,P.G.etal,Phytochemistry,1982,21(7),1643–1645;Doty,M.S.etal,Nature, 1966,211 (5052), 990)。
(22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone, English by name (22E)-3β-hydroxy-cholesta-5,22-dien-24-one, molecular formula is C27H42O2。(22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone is a kind of white powder, fusing point 114 ~ 116 DEG C。In view of the specificity of this steroidal compounds side chain, synthesis chemist adopt different synthetic methods to its carried out complete synthesis (Koch, P.etal,BulletindelaSocieteChimiquedeFrance,1983,(7-8,Pt.2),189–194;Khripach,V.A.etal,ZhurnalOrganicheskoiKhimii, 1993,29 (7), 1,368 1371;Cui Jianguo etc.,Zhongshan University's journal (natural science edition),2000,39(2),46–50;Litvinovskaya,R.P.etal,RussianJournalofOrganicChemistry(TranslationofZhurnalOrganicheskoiKhimii),2002,38(3),355–360;Morisaki,N.etal,Chemical&PharmaceuticalBulletin,2002,50(7),935–940;Cui,J.-G.etal,Steroids, 2002,67 (13-14), 1,015 1019)。This is to separate first to obtain (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone in marine algae。Further, up to now, there is not yet related compounds (22 is trans)-3 beta-hydroxies-cholesteric-5, the bioactive research report of 22-diene-24-ketone。
Summary of the invention
A kind of compound (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone of offer and its production and use is provided。This compound adopts extraction, concentrating under reduced pressure, extraction, silica gel column chromatography and gel column chromatography purification to obtain (22 is trans)-3 beta-hydroxies-cholesteric-5 from Caulerpa racemosa (Forssk) Web V. Bos, 22-diene-24-ketone, its structure measured by physicochemical constant and spectral data analysis and determine。Showing through pharmacological testing research, this compound is to A β25-35The SH-SY5Y cell of damage has significant protective effect。
Therefore, it is an object of the present invention to provide one from Caulerpa racemosa (Forssk) Web V. Bos, prepare compound (22 is trans)-3 beta-hydroxies-cholesteric-5, the method for 22-diene-24-ketone。
It is a further object to provide the purposes of compound (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone。Specifically, this compound is to A β25-35The SH-SY5Y cell of damage has significant protective effect, can be used for preparing neuroprotective and preventing the medicine of Alzheimer's disease。
First purpose according to the present invention, the present invention provides a kind of and adopts extraction, concentrating under reduced pressure, extraction, silica gel column chromatography and gel column chromatography to prepare compound (22 is trans)-3 beta-hydroxies-cholesteric-5 from Caulerpa racemosa (Forssk) Web V. Bos, the method of 22-diene-24-ketone, specifically comprises the following steps that
1) extract extractum is prepared
By freezing Caulerpa racemosa (Forssk) Web V. Bos (C.racemosa) thaw under room temperature, with ethanol seepage pressure effects three times routinely, each percolation 1 day, obtain extracting solution, extracting solution concentrating under reduced pressure is reclaimed ethanol, obtains crude extract；
2) purification is separated
(1) above-mentioned crude extract is scattered in water and becomes suspension, being extracted respectively three times with petroleum ether, ethyl acetate and n-butyl alcohol successively by suspension, gained extract concentrating under reduced pressure respectively obtains Petroleum ether extraction extractum, ethyl acetate extracts extractum and n-butanol extraction extractum；
(2) ethyl acetate extract being carried out silica gel column chromatography, with petroleum ether/acetone gradient elution, the separation component of collection utilizes silica gel thin-layer chromatography to detect, and the identical separation component of composition merges, obtain five separation components of A, B, C, D, E after concentration；Wherein component C and petroleum ether/acetone volume ratio 8:2 and 7:3 elution fraction are through SephadexLH-20 gel filtration chromatography with methylene chloride/methanol eluting, and silica gel column chromatography is with petroleum ether/dichloromethane gradient volume ratio for 7:3 eluting, and reversed phase high-performance liquid chromatography uses C18Post (10 μm, 250 × 10mm), using methanol: water=83:17(V/V) as eluent gradient eluting, flow velocity is detect under 3mL/min, 210nm wavelength], obtain compound (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone, chemical structural formula is:
In above-mentioned preparation method, in preparing extract extractum step, the described ethanol adopted that extracts is 95% ethanol。
In above-mentioned preparation method, in purification procedures, the methylene chloride/methanol eluting concentration in described SephadexLH-20 gel filtration chromatography is volume ratio 1:1。
Second purpose according to the present invention, the invention provides (22 is trans)-3 beta-hydroxies-cholesteric-5, the purposes of 22-diene-24-ketone。
The present invention adopts mtt assay to test (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone antagonism A β25-35The activity that the neuroblastoma SH-SY5Y cell survival rate caused declines。It is experimentally confirmed that (22 is trans)-3 beta-hydroxies-cholesteric-5, the SH-SY5Y cell injury in above-mentioned model is had significant protective effect by 22-diene-24-ketone。
Therefore, (22 is trans)-3 beta-hydroxies-cholesteric-5 of the present invention, 22-diene-24-ketone can be used as preparation protection neurocyte (such as, through A β25-35The SH-SY5Y cell of damage) medicine, and then can be used as preparing nerve protection medicine and prevent Alzheimer disease drug。
Accompanying drawing explanation
Fig. 1: (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone under variable concentrations to A β25-35The cell survival rate block diagram of the SH-SY5Y cell injury brought out。
Detailed description of the invention
Embodiment 1 (22 is trans)-3 β-The preparation of hydroxyl-cholesteric-5,22-diene-24-ketone
(1) by freezing Chinese Caulerpa racemosa (Forssk) Web V. Bos (C.racemosa) (picking up from Zhanjiang coastal) 5kg(dry weight) thaw under room temperature, respectively with 30L95% ethanol percolate extraction three times, each percolation 24 hours, united extraction liquid；
(2) said extracted liquid is reclaimed ethanol at temperature≤45 DEG C concentrating under reduced pressure, obtain crude extract 350g；This crude extract is scattered in water and becomes suspension, being extracted three times respectively by petroleum ether (1.5L), ethyl acetate (1.5L) and n-butyl alcohol (1L) successively by suspension, gained extract concentrating under reduced pressure respectively obtains Petroleum ether extraction extractum (38g), ethyl acetate extracts extractum (160g) and n-butanol extraction extractum (120g)；
(3) ethyl acetate extract being carried out silica gel column chromatography, with petroleum ether/acetone gradient elution, the separation component of collection utilizes silica gel thin-layer chromatography to detect, and the identical separation component of composition merges, obtain five separation components of A, B, C, D, E after concentration；Wherein component C and petroleum ether/acetone volume ratio 8:2 and 7:3 elution fraction are successively through SephadexLH-20 gel filtration chromatography (with methylene chloride/methanol eluting), silica gel column chromatography (with petroleum ether/dichloromethane gradient volume ratio for 7:3 eluting), and reversed phase high-performance liquid chromatography [uses C18Post (10 μm, 250 × 10mm), using methanol: water=83:17(V/V) as eluent gradient eluting, flow velocity is detect under 3mL/min, 210nm wavelength], obtain compound (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone。
Embodiment 2 (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone is to A β25-35The protective effect of induction SH-SY5Y cellular damage
1, laboratory sample and experimental technique
The preparation of sample solution: test sample is sterling compound (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone of preparation in above-described embodiment 1。Accurately weigh appropriate amount of sample, be configured to the solution of desired concn with DMSO, test for pharmacologically active。
The successive transfer culture of cell line and cell: active testing adopts SH-SY5Y cell line (purchased from American ATCC (Americantypeculturecollection))。With the DMEM culture medium containing 10%FBS, successive transfer culture in 37 DEG C of incubators passing into 5% carbon dioxide。
Cytoactive method of testing (mtt assay): the present invention adopts mtt assay, the tested sample of test evaluation is to A β25-35Cause the protective effect that SH-SY5Y cell viability declines。In living cells mitochondrion dehydrogenase can metabolism reduction yellow bromination 3-(4,5-dimethylthiazole)-2,5-diphenyltetrazolium bromide is hepatic water-fast first hairpin (formazan), first hairpin number can measure its trap by microplate reader and try to achieve。Owing to the amount of formazan is directly proportional to living cells, so the number of living cells can be obtained according to trap, thus understanding the sample impact on cell。
During active testing, the SH-SY5Y cell of trophophase of taking the logarithm, being configured to density by fresh DMEM culture medium is every milliliter 5 × 104The cell suspension of individual cell, is inoculated in 96 orifice plates by every hole 100 μ L, adds medicinal liquid 10 μ L/ hole after cultivating 24h, each concentration is all provided with six multiple holes, separately sets blank and model group。Cell 37 DEG C, cultivate 2h under 5% carbon dioxide conditions after, except blank group, every hole adds A β25-35(ultimate density is 1 μM) damaging cells, 37 DEG C, 5%CO2After cultivating 24h under condition, every hole adds MTT solution 10 μ L (5mg/mL), after continuing cultivation 4h, carefully removes supernatant, adds DMSO100 μ L/ hole, then survey OD by microplate reader570/630Value。The measured object protective effect to cell: survival rate %=100 × administration group OD/ matched group OD is calculated by following equation
Evaluation of result: the survival rate of sample group is more high, test sample is more strong to the protective effect of cell。
2, experimental result
(22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone is to A β25-35The protective effect Activity Results of induction SH-SY5Y cellular damage is shown in Fig. 1。(##P < 0.01 compared to blank group,**P < 0.01 is compared to model group): (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone when 10 μMs of concentration to A β25-35The SH-SY5Y cell injury caused has significant protective effect。
3, conclusion
(22 is trans)-3 beta-hydroxies-cholesteric-5, neuroblastoma cell is had significant protective effect by 22-diene-24-ketone at low concentrations, thus can be used as preparing nerve protection medicine and preventing Alzheimer disease drug。
Embodiment 3
Example 1 gained compound, adds common drug adjuvant, makes the dosage forms such as tablet, capsule, oral agents。
Claims (1)
1. one kind is extracted (22 is trans)-3 beta-hydroxies-cholesteric-5 from Caulerpa racemosa (Forssk) Web V. Bos, the method for 22-diene-24-ketone, it is characterised in that follow these steps to carry out:
(1) will thaw under freezing Chinese Caulerpa racemosa (Forssk) Web V. Bos 5kg dry weight room temperature, use 30L95% ethanol percolate extraction three times respectively, each percolation 24 hours, united extraction liquid；
(2) said extracted liquid is reclaimed ethanol at temperature 45 C concentrating under reduced pressure, obtain crude extract 350g；This crude extract is scattered in water and becomes suspension, being extracted respectively three times with petroleum ether 1.5L, ethyl acetate 1.5L and n-butyl alcohol 1L successively by suspension, gained extract concentrating under reduced pressure respectively obtains Petroleum ether extraction extractum 38g, ethyl acetate extracts extractum 160g and n-butanol extraction extractum 120g；
(3) ethyl acetate extract being carried out silica gel column chromatography, with petroleum ether/acetone gradient elution, the separation component of collection utilizes silica gel thin-layer chromatography to detect, and the identical separation component of composition merges, obtain five separation components of A, B, C, D, E after concentration；Wherein component C and petroleum ether/acetone volume ratio 8:2 and 7:3 elution fraction are successively through SephadexLH-20 gel filtration chromatography, with methylene chloride/methanol eluting；Silica gel column chromatography, with petroleum ether/dichloromethane gradient volume ratio for 7:3 eluting；Reversed phase high-performance liquid chromatography: use 10 μm, 250 × 10mmC18Post, using methanol: water=83:17V/V is as eluent gradient eluting, and flow velocity is detect under 3mL/min, 210nm wavelength, obtains compound (22 is trans)-3 beta-hydroxies-cholesteric-5,22-diene-24-ketone。
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